KR20050051862A - Sphingolipid-peg derivatives and composition for skin external use containing the derivatives - Google Patents

Abstract

The present invention relates to derivatives of sphingolipids and compositions containing the same, specifically sphingolipids selected from the group consisting of enacetylphytosphingosine, enacetylsphinginine and enacetylsphingadiene It is characterized in that the sphingolipid-polyethylene glycol polymer formed by bonding with polyethylene glycol.

The sphingolipid-polyethylene glycol polymer according to the present invention inhibits tyrosinase enzyme activity and inhibits melanin biosynthesis, thereby preventing and treating skin pigmentary symptoms such as blemishes, freckles, senile plaques, and skin pigmentation hyperdeposition. Can be. Furthermore, the sphingolipid-polyethylene glycol polymer according to the present invention can be used in whitening cosmetics and pharmaceuticals. The sphingolipid-polyethylene glycol polymer according to the present invention exhibits an improved whitening effect as sphingolipids are combined with polyethyleneglycol. In addition, the solubility of sphingolipids is improved and the toxicity is reduced, thereby greatly extending the range of its use. There is this.

Description

Sphingolipid-PEG derivatives and composition for skin external use containing the derivatives}

The present invention relates to derivatives of sphingolipids and compositions containing them. More specifically, sphingolipid-polyethyleneglycol polymer formed by combining sphingolipids selected from the group consisting of enacetylphytosphingosine, enacetylsphinginine and enacetylsphingadiene with polyethylene glycol and the skin of the polymer It relates to whitening uses.

Hyperpigmentation on the skin is a serious mental burden from a cosmetic point of view and, in severe cases, severely disrupts social activities. In addition, the development of whitening agents has been actively carried out in accordance with the social changes that white and fine skin is the standard of beauty.

Recently, research on the whitening agent is progressing to control various factors involved in the melanin synthesis process. The first step is to control the cytokines and hormones from the sun bloc, to control the internal signal of melanocytes, to inhibit the activity of tyrosinase enzymes, and to move the melanosomes. Progress has been made in a number of ways, such as inhibiting and melanin-depleted skin cells.

Sphingolipids are well known as substances that play an important role in cell growth, differentiation and death by participating in intracellular signaling. Ceramide is a lipid that is most present in the skin and inhibits evaporation of water from the skin, thereby preventing aging and skin diseases. In addition, phytosphingosine and its acetylated derivatives, which are sphingolipid long chain bases, are known to have excellent antibacterial and anti-inflammatory effects and are also involved in the biosynthesis of ceramide in the skin. However, although there have been many studies on ceramide and sphingolipid derivatives in keratinocytes, many studies on melanin synthesis have not been conducted.

Kim (Kim, Dong-Seok, Cellular signaling 14 (2002) 779 ~ 785) first conducted a study involving melanin synthesis using sphingolipid derivatives, and ceramide derivatives (C2 ceramides) were compared to kojic acid at concentrations of 1 to 10 uM. It has been reported to show an excellent melanin synthesis inhibitory effect. It has also been reported that sphingosine-1-phosphate inhibits melanin synthesis through regulation of the signaling process of melanin synthesis. On the other hand, Doosan Corporation has applied for phytosphingosine derivative N-acetylphytosphingosine to prove that it has a whitening effect (Patent Application No. 2002-72043).

On the other hand, sphingolipids have a very low solubility, which makes them difficult to use in various formulations.

Polyethylene glycol is a substance having the structure of HO- (CH ₂ CH ₂ O) nCH ₂ CH ₂ -OH and is often used in foods and cosmetics. Has been used. Polyethyleneglycol is a non-toxic polymer, has good solubility, is not antigenic in the human body, and is easily removed from the body. Polyethylene glycol has a hydroxy group at both ends, so in actual PEGylation, monomethoxy polyethylene glycol (B ₃ ) in which one end is blocked (CH ₃ O- (CH ₂ CH) ₂ O) nCH ₂ CH ₂ -OH) is used a lot and the molecular weight is up to 30,000 Dalton (Dalton). In order to bind monomethoxy polyethyleneglycol to a medicament, the hydroxyl group of the methoxy unbonded terminal is activated and bound using various chemicals.

Lewis S. L et al. (U.S. Pat. No. 5,820,873, 1995) attempted to synthesize derivatives of polyethylene glycol-ceramide and apply them to liposomes.

It is an object of the present invention to prevent melanin pigmentation by inhibiting tyrosinase activity using sphingolipid derivatives, thereby preventing skin pigmentation symptoms such as blemishes, freckles, senile plaques, and skin pigmentation hyperdeposition. It aims to do it. It is also an object of the present invention to provide an external composition for skin having such an effect.

In order to achieve the above object, the sphingolipid-polyethylene glycol polymer according to the present invention, N-acetylphytosphingosine (N-acetylphytosphingosine), N-acetylsphinganine and N-acetylsphingadiene ( N-acetylsphingadiene) is characterized in that the sphingolipid selected from the group consisting of polyethylene glycol formed.

The sphingolipid-polyethylene glycol polymer according to the present invention described above is characterized in that it is any one of the compounds of the following general formulas (1) to (3).

Wherein, R ₁ is independently hydrogen, an alkynyl group, an acyl group or an aryl group of C _1-40 alkyl group, a C _1-40 alkenyl group, C _1-40,

In the case of an acyl group (Cor ₂ ), R ₂ is an alkyl group, an alkenyl group, an alkynyl group, or an aryl group.

X is NR _3- , O-, -S- or X ₁ -alk-X ₂ , wherein R ₃ is hydrogen, an alkyl, acyl or aryl group of C _1-6 , and X ₁ and X ₂ are independently Amino group, amido group, carboxyl group, carbamate group, carbonyl group, urea or phosphoro (phosphoro-), and alk is C _1-6 alkynene.

In the sphingolipid-polyethylene glycol polymer according to the present invention, the polyethylene glycol is characterized in that the molecular weight of 550 to 10,000, more preferably the polyethylene glycol has a molecular weight of 700 to 5,000, the terminal is a methoxy group It is characterized by being substituted by.

In addition, the external preparation composition for skin according to the present invention is characterized in that it contains the sphingolipid-polyethylene glycol polymer as an active ingredient.

The external preparation composition for skin according to the present invention is characterized by inhibiting tyrosinase activity.

The external preparation composition for skin according to the present invention described above is for whitening.

The external preparation composition for skin according to the present invention described above is a cosmetic composition.

The external preparation composition for skin according to the present invention is characterized in that it contains 0.005 to 10% by weight of the sphingolipid-polyethylene glycol polymer.

In addition, the method for inhibiting tyrosinase activity according to the present invention is characterized by using the sphingolipid-polyethylene glycol polymer as described above.

In addition, the method for inhibiting melanin synthesis according to the present invention is characterized by using the sphingolipid-polyethylene glycol polymer as described above.

Hereinafter, the present invention will be described in more detail.

The present invention is characterized by increasing the whitening effect by binding polyethylene glycol to sphingolipids. The inventors have found that sphingolipid-polyethyleneglycol polymers show an excellent whitening effect compared to sphingolipid itself.

Representative examples of sphingolipid-polyethylene glycol polymers according to the present invention are those wherein R ₁ is independently hydrogen, an alkyl group, or an acyl group, X is succinate, and polyethylene glycol Has its own weight ranging from 750 to 5000 and its end is replaced by a methoxy group.

The sphingolipid-polyethylene glycol polymer according to the present invention can be prepared by reacting with polyethylene glycol preactivated in a solvent in the presence of a catalyst.

The manufacturing method will be described in more detail as follows. First, polyethylene glycol is activated. Succinic anhydride is added to polyethylene glycol in the presence of an organic solvent to convert alcohol groups to carboxyl groups. Triethylamine and p-Toluenesulfonylchloride were activated on the carboxyl-substituted polyethylene glycol to activate the acid moiety, and a small amount of sphingolipid was added to the activated solution for 24 hours. Complete.

The compound prepared by the above-described method is extracted with an organic solvent such as chloroform or a mixed solvent of chloroform / metalol and purified by adsorption chromatography on silica gel.

The composition according to the present invention is characterized in that it is an external composition for skin, and in particular, may be a cosmetic composition, and in particular, it is particularly a soft cosmetics, nourishing cosmetics, massage creams, nutrition creams, gels, packs or cosmetic formulations of the skin adhesive type. Do.

The composition according to the present invention is characterized in that it contains 0.005 to 10% by weight of sphingolipid-polyethylene glycol polymer. It should be 0.005% by weight or more to achieve the whitening effect to be achieved by the present invention, and 10% by weight or less of the formulation stability is excellent.

In the sphingolipid-polyethylene glycol polymer according to the present invention, the polyethylene glycol preferably has a molecular weight of 550 to 10,000, more preferably 700 to 5,000. The molecular weight range is the most optimal range for achieving the purpose of bonding polyethylene glycol in the present invention.

The compositions according to the invention can also be in transdermal dosage forms such as lotions, ointments, gels, creams, patches or sprays.

Human skin color is determined by several skin components. Of these, melanin produced by the melanocytes of the skin (Melanin) plays the most important role. The melanocytes of the skin are regulated to have a certain degree of skin color by genetic traits according to race. However, when stimulated with melanin-synthesizing agents such as UV rays, stress, cytokines, and hormones (Melanocyte-stumulating Hormone (MSH)), the synthesis of melanin increases and the synthesized melanin is synthesized by melanosomes. It is transported to keratinocytes and eventually undergoes desquamation from the skin.

In general, the change in the skin color temporarily returns to the previous skin color with the above mechanism, but the skin color may be irreversibly changed when exposed to sunlight for a long time.

Melanin is synthesized in melanosomes in melanocytes, which are derived from the endoplasmic reticulum (Endoplasmin reticulum) and melanin accumulates regularly by the structure inside melanosomes. Tyrosinase, an enzyme that converts tyrosine to melanin, is produced in the Golgi apparatus and then migrated to melanosomes. In melanosomes before maturation, the synthesis of melanin begins by tyrosine oxidizing by tyrosinase. Melanosomes, which have begun to synthesize melanin, are then matured in about four stages to complete the production of mature melanosomes. The mature melanosomes migrate to the surrounding keratinocytes and the skin color is determined by the number, size, and distribution of melanin in the keratinocytes. Melanosomes migrate from the basal layer of the epidermis to keratinocytes, and to the stratum corneum, protecting keratinocytes. Melanosomes disintegrate during migration, but melanin remains intact and eventually leaves the skin.

Tyrosinase, which plays the most important role in melanin biosynthesis, is synthesized in Golgi and then migrated to melanosomes through glycosylation, and then activated by phosphorylation after physphorylation. Is switched.

Synthesis of melanin begins when activated tyrosinase causes oxidation by tyrosine as a substrate. Tyrosine is converted to dopa (DOPA: dihydroxyphenylalanine) by tyrosinase, and back to dopaquinone (DOPA quinone) by tyrosinase. Dopaquinone, on the one hand, binds to glutathione or cysteine and is converted to phenomelanin via Cysteinyl DOPA. On the other hand, dopaquinone is converted to dopachrome, and then through dihydroxyindole (5,6-Dihydroxyindole), and again by the action of tyrosinase (Indole-5,6-quinone) It is then converted to eumelanin. Tyrosinase is known as the most important enzyme of melanin synthesis process by participating in not only the initial stage but also the final stage of melanin synthesis reaction, and the substance with many melanin formation inhibitory effects is aimed at the substance that inhibits the activity of tyrosinase. It was developed as, the composition according to the invention is also characterized by containing sphingolipids, such as enacetyl phytosphingosine to inhibit the activity of tyrosinase.

Eumelanin and peomelanin synthesized in melanocytes are present in different ratios according to race and human body parts, thereby causing a difference in skin color. Eumelanin is black and brown in color, and pheomelanin is orange and is synthesized from two proteins, tyrosine and cysteine. Melanin protects the skin from UV rays, protects the sub-dermal dermal organs, and protects proteins and nucleic acids by trapping free radicals and free radicals generated in the skin. However, blemishes, freckles, and skin pigmentation are caused by abnormal production of melanin pigment, which functions as described above, and deposition on the skin.

When the composition according to the invention is a pharmaceutical composition of a transdermal dosage form, the preferred dosage is 0.001 to 1000 mg / kg body weight twice daily, based on a composition containing 0.005 to 10% by weight of the enacetylphytosphingosine-polyethylene glycol polymer. It is preferable to administer.

Hereinafter, the present invention will be described in more detail with reference to a preferred embodiment of the present invention, but the present invention is not limited by the following examples.

Example

Preparation Example 1 Preparation of Sphingolipid-Polyethylene Glycol Polymer according to the Present Invention

5.0 g (0.0067 mol) of methoxy polyethylene glycol was dissolved in a pyridine solvent under nitrogen gas, and 6.67 g (0.067 mol) of succinic anhydride was slowly added thereto, and the reaction was performed at room temperature for 24 hours while blocking light. After the reaction was completed, the pyridine was converted to the salt form by adding hydrochloric acid to pH 2, and extracted by removing chloroform and water. Excess succinic anhydride was likewise extracted and removed from the water layer at this time. 1 g (0.0011 mol) of the methoxy polyethylene glycol-carboxylic acid thus obtained was dissolved in dichloromethane solvent under nitrogen gas, and 0.17 ml (0.0017 mol) of triethylamine and 2.11 g (0.011 mol) of paratoluenesulfonyl chloride were used. After activating the carboxyl group, 0.22 g (0.0011 mol) of acetylphytosphingosine was slowly added, and the reaction was allowed to proceed for 24 hours under warming. After completion of the reaction, the reaction was stopped by extraction with distilled water and purified by adsorption chromatography using silica gel to obtain an enacetylphytosphingosine-polyethylene glycol polymer according to the present invention. Succinic anhydride reacted with succinic anhydride to confirm the introduction of succinic acid into methoxypolyethylene glycol (δ = 2.6 ppm, t, 4H) and finally methylene of acetylphytosphingosine (δ = 1.2 ppm, m). , 48H) and (δ = 0.9 ppm, t, 6H) and ethoxy (δ = 3.6 ppm, m, 64H) and methoxy (δ = 3.3 ppm, s, 3H) It was confirmed that the present derivative was synthesized by combining polyethylene glycol and enacetyl phytosphingosine.

Examples 1 to 6: Preparation of Cream Composition

The present inventors prepared the preferred embodiments according to the present invention as follows. Examples 1 to 6 below are cream compositions each containing an enacetyl phytosphingosine-polyethylene glycol polymer at different concentrations. Meanwhile, a cream composition according to Comparative Example 1 was prepared as a negative control for comparison with Examples 1 to 6.

ingredient(%) Comparative Example 1 Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Enacetyl Phytosphingosine-Polyethylene Glycol Polymer - 0.05 0.1 0.2 0.5 - - N-acetylsphinginine-polyethylene glycol polymer - - - - - 0.2 - N-Acetyl Spingadiene-Polyethylene Glycol Polymer - - - - - - 0.2 cholesterol One One One One One One One Free fatty acids One One One One One One One Lecithin 0.5 0.5 0.5 0.5 0.5 0.5 0.5 glycerin 5 5 5 5 5 5 5 1,3-butylene glycol 2 2 2 2 2 2 2 Carbopol 940 0.2 0.2 0.2 0.2 0.2 0.2 0.2 TEA 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Germall 115 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Phytoscualan 15 15 15 15 15 15 15 Etc 35 35 35 35 35 35 35 water to 100 to 100 to 100 to 100 to 100 to 100 to 100

The present inventors conducted the following experiment to demonstrate the whitening effect of the sphingolipid-polyethylene glycol polymer according to the present invention.

Experimental Example 1 Inhibitory Effect of Sphingolipid-Polyethylene Glycol Polymer on Melanin Production

Enacetyl Phytosphingosine-Polyethylene Glycol Polymer, Enacetylsphinginine-Polyethylene Glycol Polymer, Enacetylsphingadiene-Polyethylene Glycol Polymer, Enacetyl Physphingosine, Enacetyl Spingane, Enacetylsphingadiene, and Kojic Acid The melanin production inhibitory effect of was measured. The above material was dissolved in DMSO (dimethyl sulfoxide) when used. In addition, the cell line used in the present invention, B16F10 Melanoma, a mouse skin cancer cell, in a DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS (Fetal bovine serum) and antibiotics (antibiotics) at a temperature of 37 ℃ and 5% CO was used and cultured in a CO ₂ incubator which _second air is supplied with a.

The measurement method of melanin content is as follows. B16F10 mouse melanoma cells were seeded in 24-well multi-plates at 2 × 10 ³ cells / well and incubated in 37 ° C., 5% CO ₂ incubator for 24 hours. After pre-culture, the test materials were diluted in Dulbecco's Modified Eagle Medium (DMEM) containing no serum, and treated with cells by concentration, followed by incubation for 96 hours. After washing with PBS, 0.85N KOH was added, dissolved in ultrasonic wave, the absorbance was measured at 475 nm, and the total amount of melanin was measured. Meanwhile, the fractions of eumelanin and peomelanin were separated by high-speed chromatography, and the production rate for the control group. It was determined in terms of. The results are as shown in the table below. The following results are melanin production rates, which represent% production rate for untreated cells.

sample Melanin production rate (%) 100 μM 10 μM Untreated 100 100 Enacetylphytosphingosine - 17 Enacetyl Phytosphingosine-Polyethylene Glycol Polymer - 9 N-acetylsphinginine - 23 N-acetylsphinginine-polyethylene glycol polymer - 14 N-acetyl spingadiene - 35 N-Acetyl Spingadiene-Polyethylene Glycol Polymer - 28 Kojic acid 10 90

As shown in the above results, the concentration that inhibits melanin synthesis of about 90% in all samples should be treated with about 100 uM for kojic acid, whereas about 1 for sphingolipids or sphingolipid-polyethylene glycol polymers. Almost 10% of melanin synthesis was inhibited at 10 uM. Although not described in this example, it was confirmed that arbutin had the same level of inhibitory effect at a concentration of 10 times kojic acid and 100 times of the substance of the present invention. In addition, as shown in the table, sphingolipid-polyethylene glycol polymer was found to be more than twice as effective in inhibiting melanin synthesis than sphingolipid.

 On the other hand, the present inventors photographed the state of collecting the cell line used in this experiment by centrifugation. The results are shown in FIG. In Figure 1, the lower color indicates the degree of melanin synthesis, it can be seen that in the case of enacetyl phytosphingosine melanin production is suppressed, the color of the cell mass does not turn black. It was found that the color was much lighter than the enacetyl phytosphingosine, the melanin production inhibitory effect is more excellent.

Experimental Example 2: Inhibition of Tyrosinase Activity of Enacetyl Phytosphingosine-Polyethylene Glycol Polymer

The present inventors experimented as follows to determine the effect on the tyrosinase activity of the enacetylphytosphingosine-polyethylene glycol polymer.

In this experimental example, as in Experiment 1, the enacetyl phytosphingosine-polyethylene glycol polymer was dissolved in DMSO (dimethyl sulfoxide). In addition, the cell line used in the present invention, B16F10 melanoma, a mouse skin cancer cell, using Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS (Fetal bovine serum) and antibiotics, contains 37% temperature and 5% CO ₂ . It was used by culturing in a CO ₂ incubator supplied with air. Tyrosinase activity inhibition is measured as follows. B16F10 mouse melanoma cells were seeded in 96-well multi-plates at 1 × 10 ⁴ cells / well and incubated for 24 hours in 37 ° C., 5% CO ₂ incubator. After pre-incubation, the test materials were diluted in Dulbecco's Modified Eagle Medium (DMEM) containing no serum, treated with cells by concentration, incubated for 96 hours, and washed twice with PBS. 1% Triton X-100 / PBS was added thereto. After dissolving with ultrasonic wave, 5 μl of 10 mM L-Dopa prepared at 37 ° C. was reacted for 30 minutes at 37 ° C., and absorbance was measured at 475 nm. The results are shown in Figure 2b. The results in Figure 2 are tyrosinase activity, indicating activity (%) against untreated cells. 2A shows a standard curve of tyrosinase.

As shown in Figure 2b it can be seen that the effect of inhibiting tyrosinase enzyme activity close to about 50% at a concentration of about 10uM of the enacetyl phytosphingosine-polyethylene glycol polymer. This is similar to the results of the melanin synthesis experiment, the concentration of 1/10 of kojic acid and close to 1/100 of arbutin, it can be seen that the inhibitory effect is excellent at the same concentration compared to arbutin or kojic acid.

Experimental Example 4: Whitening Effect Test on Human Skin of Cream Composition According to the Present Invention

The inventors carried out this experiment to confirm whether the composition according to the invention has a whitening effect on the actual human skin. The whitening effect of the cream compositions of Examples 1 to 6 was measured, and the whitening effect of Comparative Example 1 was also measured.

Twenty healthy men and women were placed on the subject's arm with an opaque tape with a hole of 1.5 cm in diameter and irradiated with UVB (1.5-2 times the minimum erythema dose) of each subject. After inducing blackening of the skin, the cream composition according to Examples 1 to 6 and the cream composition according to Comparative Example 1 were applied, respectively, and two months later, the contrast of the skin was measured using a color difference meter. Each test substance was corrected twice daily, morning and evening.

The determination of the effect was determined by obtaining the L value representing the contrast of the skin.

△ L = L value on the last day of test substance application-L value before test substance application

The results are shown in Table 3 below.

Comparative Example 1 Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Whitening effect 1.5 2.32 3.12 3.55 4.21 3.45 3.37

As shown in the above results, the sphingolipid-polyethylene glycol polymer according to the present invention was confirmed that the whitening effect is excellent depending on the concentration.

The sphingolipid-polyethylene glycol polymer according to the present invention inhibits the activity of tyrosinase, thereby inhibiting the synthesis of melanin and thereby preventing skin pigmentation symptoms such as blemishes, freckles, senile plaques, and skin pigmentation hyperdeposition. have. Furthermore, the materials of the present invention can be used in cosmetics and pharmaceuticals.

1 is a photograph showing the melanin production inhibitory effect of sphingolipid-polyethylene glycol polymer according to the present invention.

2A is a graph showing a standard curve of change in OD value (475 nm) according to the amount of tyrosinase.

Figure 2b is a graph showing the inhibitory effect of tyrosinase activity of sphingolipid-polyethylene glycol polymer according to the present invention.

Claims (11)

A sphingolipid-polyethyleneglycol polymer formed by combining sphingolipids selected from the group consisting of enacetylphytosphingosine, enacetylsphinginine and enacetylsphingadiene with polyethylene glycol. The method of claim 1, The sphingolipid-polyethylene glycol polymer, Sphingolipid-polyethylene glycol polymer, characterized in that any one of the compounds of formulas (1) to (3). [Formula 1] [Formula 2] [Formula 3] Wherein, R ₁ is independently hydrogen, an alkynyl group, an acyl group or an aryl group of C _1-40 alkyl group, a C _1-40 alkenyl group, C _1-40, In the case of an acyl group (Cor ₂ ), R ₂ is an alkyl group, an alkenyl group, an alkynyl group, or an aryl group. X is NR _3- , O-, -S- or X ₁ -alk-X ₂ , wherein R ₃ is hydrogen, an alkyl, acyl or aryl group of C _1-6 , and X ₁ and X ₂ are independently Amino group, amido group, carboxyl group, carbamate group, carbonyl group, urea or phosphoro (phosphoro-), and alk is C _1-6 alkynene. The sphingolipid-polyethylene glycol polymer according to claim 2, wherein the polyethylene glycol has a molecular weight of 550 to 10,000. The sphingolipid-polyethylene glycol polymer according to claim 3, wherein the polyethylene glycol has a molecular weight of 700 to 5,000 and a terminal is substituted with a methoxy group. A topical skin composition comprising the sphingolipid-polyethylene glycol polymer according to any one of claims 1 to 4 as an active ingredient. 6. The composition for external application for skin according to claim 5, wherein the composition inhibits tyrosinase activity. 6. The composition for external application for skin according to claim 5, wherein the composition is for whitening. The composition for applying the external of the skin according to claim 5, wherein the composition is a cosmetic composition. 6. The composition for external application for skin according to claim 5, wherein the composition contains 0.005 to 10% by weight of sphingolipid-polyethylene glycol polymer. A method of inhibiting tyrosinase activity using the sphingolipid-polyethylene glycol polymer according to any one of claims 1 to 4. A method of inhibiting melanin synthesis using the sphingolipid-polyethylene glycol polymer according to any one of claims 1 to 4.